*3.4. Functional Annotation and Classification of K133WT and K133AS-R Unigenes*

Additional validation, functional annotation, and classification of K133WT and K133AS-R unigenes derived from reference-based assembly data was performed as described in the methodology section. Out of 7671 genes retrieved in K133WT and 7792 genes in K133AS-R, a total of 7652 (99.75%) in K133WT and 7778 (99.82%) genes were found with BLAST hits. K133AS-R unigenes were annotated with at least one biological term from GO information, while the remaining 19 genes in K133WT and 14 in K133AS-R did not result in any BLAST hit. Species distribution analysis based on BLASTx results with BLAST hit sharing showed high sequence similarity with *L. donovani* and *L. infantum* sequences (Figure 4A,B). In K133WT, 2925 GO terms were allocated to biological processes, 2987 terms to cellular components, and 3152 GO terms to molecular functions, while in K133AS-R, 2942 GO terms were assigned to biological processes, 3005 terms to cellular components, and 3178 GO terms to molecular functions. Within the biological process category, cellular metabolism, cellular component organization, or biogenesis was most abundant. Within the cellular component category, GO terms corresponded to the cell and organelle part, membrane part, and protein-containing complexes (Figure 4C,D). Under molecular function category, GO terms mostly corresponded to different catalytic, binding, and transporter activity, which were abundant among unigenes [52].

**Figure 4.** *Cont*.

**Figure 4.** Characterization of K133WT and K133AS-R unigenes based on an NCBI non redundant (Nr) protein database search. (**A**) Species distribution of the top Blast hits for the K133WT assembled unigenes and (**B**) Species distribution of the top Blast hits for the K133AS-R assembled unigenes with a cutoff E-value of 10−05. Gene Ontology (GO) annotation for all the assembled unigenes in K133WT (**C**) and K133AS-R (**D**) GO-terms were assigned to functionally annotate the genes based on BLAST search results using the Blast2GO program (Biobam BioInformatics, Valencia, Spain). The results were classified based in three functional categories, Green bar represents biological function (BF); Blue: molecular function (MF); and Yellow: cellular component (CC).
